Although infection with Mycobacterium tuberculosis (MTb) is the largest cause of pulmonary disease and death in HIV patients globally, little is known about how TB co-infection impacts long-term antiretroviral (ART)- mediated immune reconstitution. The Cambodian Early vs. Late Introduction of Antiretrovirals (CAMELIA) randomized clinical trial showed that early initiation of ART at 2 weeks after TB therapy initiation versus late initiation of ART at 8 weeks in severely immunocompromised TB+/HIV+ patients (median CD4=24/mm3), resulted in a significant (34%) decrease in mortality?a survival benefit that persisted for at least 3 years after the timing intervention. In a scientific sub-study nested within the CAMELIA trial, we have discovered that active TB disease in these highly immunosuppressed TB+/HIV+ patients led to significantly greater pre-ART levels of several pro-inflammatory cytokines and greater pre-ART frequencies of activated CD4+ and CD8+ T cells, and significantly lower pre-ART frequencies of CD28+CD8+ and ICOS+CD4+ T cells, as compared to TB-/HIV+ patients. Moreover, these differences in T cell subset frequencies persisted for at least 8 months after treatment initiation and 2 months after TB cure. Furthermore, we have demonstrated that TB-associated immune reconstitution syndrome (TB-IRIS), which occurred 2.6-fold more frequently in the early CAMELIA treatment arm, results in profound changes in the T cell compartment. For example, TB-IRIS patients exhibited a significant post-ART expansion of (CD62L-CD45RA-) effector memory CD4+T cells and a decrease in (CD62L+CD45RA-) central memory CD4+T cells as compared to non-TB-IRIS patients. We have also found that a Th1-like CD4+ T cell subset that is CXCR3+CCR6+ and thought to mediate a large portion of anti-TB responses was present at significantly elevated frequencies pre-ART in TB-IRIS patients, and the frequency of these cells remained elevated for at least 8 months after treatment onset. Based on our preliminary data, we hypothesize that TB co-infection exacerbates HIV-induced premature aging of the immune system, leading to long-term consequences and poor recall responses. In Aim 1 we will investigate immunosenescent and methylomic signatures in pre-treatment archived samples from TB+/HIV+ CAMELIA and TB-/HIV+ and TB+ /HIV- patients, and in freshly isolated samples from the same patients >5 years after TB treatment and ART initiation. We further hypothesize that within the TB+/HIV+ group the subset of patients who experienced TB- IRIS possessed superior anti-MTb immunity, which was amplified once ART was initiated. In Aim 2 we will compare CXCR3+CCR6+CD4+ T cells in TB-IRIS versus non-TB-IRIS patients to determine their anti-MTb function. Findings from these experiments will provide critical insights into how TB co-infection in HIV+ patients affects phenotypic and epigenetic features of the immune system prior to and following ART-mediated immune reconstitution, and they will elucidate the long-term consequences of TB-IRIS on functional MTb-specific T cell responses that were associated with successful ART-mediated immune reconstitution and TB cure.